While differences between vascular and phagocytic NADPH oxidase are yet to be identified, reactive oxygen species (ROS) have emerged as important regulatory molecules which function as critical second messengers modulating signal transduction pathways involved in endothelial cell growth, migration, adhesion and barrier function. While actin cytoskeleton has been implicated in phagocytosis and oxidative burst in neutrophils, very little is known regarding the role of actin cytoskeleton in endothelial NAD[P]H oxidase assembly and activation. Our preliminary studies demonstrate that acute hyperoxia-mediated activation of NAD[P]H oxidase and enhanced production of superoxide is regulated by cortical actin cytoskeleton rearrangement, phosphorylated myosin light chain, small G-proteins and phosphatidic acid. We hypothesize that acute hyperoxia-induced endothelial NAD[P]H oxidase activation is mediated by signal transduction pathways regulating cortical actin cytoskeleton remodeling and by facilitating the assembly of the oxidase sub-components. The following specific aims will investigate the relationship between acute hyperoxia, actin cytoskeletal remodeling and NAD[P]H oxidase assembly and activation in human pulmonary artery endothelial cells in the absence of cytotoxicity. SA#1: Will investigate pathways linking actin cytoskeleton remodeling in NAD[P]H oxidase activation; SA#2: Will characterize the role of myosin light-chain phosphorylation in NAD[P]H oxidase activation; SA#3: Will define the role of cortactin and coronin in endothelial NAD[P]H oxidase activation and SA#4: Will characterize the role of phospholipase D / phosphatidic acid signaling in endothelial NAD[P]H oxidase activation. An understanding of the role of actin cytoskeletal remodeling in NAD[P]H oxidase assembly and activation will allow development of targeted therapies to modulate ROS-induced signaling and prevent lung injury and endothelial dysfunction.